Great promise still exists for the use of gene therapy in cancer treatment. The most direct approach is expression of toxic or immunostimulatory genes within tumor cells in vivo. This requires an efficient and targeted vector system. Replication-selective vectors provide the most promising means of achieving this goal. While numerous tumor cell active replicating vectors have been studied and some are in clinical trial, there is significant room for improvement of these vectors. We have explored pox viruses as tumor-selective replicating vectors for tumor directed gene delivery and direct tumor destruction. The pox viruses have the advantage of being extremely efficient in vivo. We have intentionally utilized an efficient strain of vaccinia virus (WR strain), which can rapidly spread through and destroy tissue. We have performed mutations of this vector to make it selective for tumors. Remarkably a systemic injection of this virus can infect tumor cells and directly destroy tumors up to 1 cm in size in our preliminary studies. While we are currently exploring clinical trials with this vector we realize there are many ways to improve this system. The proposed research will address the limitations of this vector to continue the preclinical development for cancer gene therapy. Ultimately, the vector can be utilized as a direct "oncolytic" virus to express toxic genes within tumors, or to take advantage of the tumor environment to express immunostimulatory genes for development of systemic immunity against the tumor. The aims of this study are 1.) Enhance tumor selectivity and safety by combining deletions of vaccinia serpins with TK and VGF, and define the mechanism of tumor selective replication. 2: Develop a tetracycline inducible gene expression system with tight in vivo regulation and creation of a "shut off" switch for viral replication. 3: Immunosuppress to enhance the safety and efficacy of Vaccinia treatment and to allow successful treatment of the pre-immunized host. The research will not only help develop vaccinia virus as a vector but should have implications for other replicating vectors used for cancer gene therapy. Our goal is to explore any advances over our current vector in clinical trials in metastatic gastrointestinal cancers.